Warm and cold parental reproductive environments affect seed properties, fitness, and cold responsiveness in Arabidopsis thaliana progenies.

Conditions in the parental environment during reproduction can affect the performance of the progenies. The goals of this study were to investigate whether warm or cold temperatures in the parental environment during flowering and seed development affect Arabidopsis thaliana seed properties, growth performance, reproduction and stress tolerance of the progenies, and to find candidate genes for progeny-related differences in stress responsiveness. Parental plants were raised at 20 degrees C and maintained from bolting to seed maturity at warm (25 degrees C) or cold (15 degrees C) temperatures. Analysis of seed properties revealed significant increases in nitrogen in seeds from warm temperature and significant increases in lipids and in the ratio of alpha-linolenic to oleic acid in seeds from the cold parental environment. Progenies of the warm parental environment showed faster germination rates, faster root elongation growth, higher leaf biomass and increased seed production at various temperatures compared with those from the cold parental environment. This indicates that under stable environmental conditions, progenies from warm parental environments had a clear adaptive advantage over those from cold parental environments. This parental effect was presumably transmitted by the higher nitrogen content of the seeds developed in warm conditions. When offspring from parents grown at different temperatures were exposed to chilling or freezing stress, photosynthetic yield recovered faster in progenies originating from cold parental environments. Cold acclimation involved up-regulation of transcripts of flavanone 3-hydroxylase (F3H) and pseudo response regulator 9 (PRR9) and down-regulation of growth-associated transcription factors (TFs) NAP and AP2domain containing RAP2.3. NAP, a regulator of senescence, and PRR9, a temperature-sensitive modulator of the circadian clock, were probably involved in mediating parent-of-origin effects, because they showed progeny-related expression differences under chilling. Because low temperatures also delay senescence, cold responsiveness of NAP suggests that this factor is linked with the regulatory network that is important for environmental acclimation of plants.

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells.

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P(TF), was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P(TF) to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P(NSE)) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.

Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-repressible regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes.

The chimeric transcriptional activator TGV mediates dexamethasone (dx)-inducible and tetracycline (tc)-repressible transgene expression in tobacco (dx-on/ tc-off system). The expression profiles of four different synthetic target promoters, comprising multiple TGV binding sites upstream of a core promoter, were characterised using the sensitive luciferase assay. Induction factors of over 1,000 were measured in roots and leaves of over 30% of the transgenic plants, irrespective of the promoter used. Promoters PTF and PTax, which carry the -48 to +1 region of the Cauliflower Mosaic Virus 35S promoter, showed higher expression levels in both the uninduced and induced states than PTop10 and PTFM, which harbour several point mutations in this region. Moreover, PTax expressed higher background activities than PTF, indicating that the sequence of the synthetic regulatory region can influence background levels. The usefulness of the dx-on/tc-off system for experiments addressing gene function was demonstrated by using it to control the expression of isopentenyl transferase. This enzyme catalyses the rate-limiting step in cytokinin biosynthesis and causes phenotypic effects even at low expression levels. Only dx-induced transgenic plants displayed phenotypic alterations indicative for increased cytokinin synthesis (e.g. outgrowth of lateral buds). Simultaneous treatment of selected buds with the antiinducer tc suppressed bud growth. This result suggests that cytokinins cannot serve as mobile signals to elicit the release of apical dominance in tissues compromised for enhanced cytokinin synthesis.

Phytochrome A resets the circadian clock and delays tuber formation under long days in potato.

Transgenic potatoes (Solanum tuberosum) with either increased (sense transformants) or reduced (antisense transformants) phytochrome A (phyA) levels were used, in combination with specific light treatments, to investigate the involvement of phyA in the perception of signals that entrain the circadian clock. Far-red or far-red plus red light treatments given during the night reset the circadian rhythm of leaf movements in wild-type plants and phyA over-expressors, but had little effect in phyA under-expressors. Far-red light was also able to reset the rhythm of leaf movement in wild-type Arabidopsis thaliana but was not effective in mutants without phyA. Blue light was necessary to reset the rhythm in phyA-deficient potato plants. Resetting of the rhythm by far-red plus red light was only slightly affected in transgenic plants with reduced levels of phytochrome B. The production of tubers was delayed by day extensions with far-red plus red light, but this effect was reduced in transgenic lines deficient in phyA. We conclude that phyA is involved in resetting the circadian clock controlling leaf movements and in photoperiod sensing in light-grown potato plants.

Tobacco TGA factors differ with respect to interaction with NPR1, activation potential and DNA-binding properties.

In higher plants, as-1-like cis elements mediate auxin- and salicylic acid-inducible transcription. Originally found in viral and T-DNA promoters, they are also functional elements of plant promoters activated during the defence response against pathogens. Tobacco bZIP transcription factor TGA1a was the first recombinant protein shown to bind to as-1. cDNAs for two novel tobacco as-1-binding bZIP proteins (TGA2.1 and TGA2.2) were isolated. Revealing a high degree of amino acid identity in the bZIP domain (89%) and the C-terminus (79%), the two TGA2 factors differ remarkably with respect to the length of the N-terminus (170 amino acids in TGA2.1 versus 43 amino acids in TGA2.2). TGA2.1 and TGA2.2, but not TGA1a, interacted with ankyrin repeat protein NPR1, a central activator of the plant defence response. In contrast, TGA2.1 and TGA1a, but not TGA2.2, functioned as transcriptional activators in yeast. Apart from conferring transcriptional activation, the N-terminal domain of TGA2.1 led to reduced in vitro as-1-binding activity and almost completely abolished binding to one half site of this bifunctional element. When being part of a heterodimer with TGA2.2, TGA2.1 was efficiently recruited to a single half site, though double occupancy of the element was still preferred. In contrast, TGA1a preferred to bind to only one palindrome, a feature that was also maintained in heterodimers between TGA1a and TGA2.1 or TGA2.2.

Tobacco transcription factor TGA2.2 is the main component of as-1-binding factor ASF-1 and is involved in salicylic acid- and auxin-inducible expression of as-1-containing target promoters.

In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation. Originally found in promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack. Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid response protein (SARP) bind specifically to as-1. Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP. Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable. Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2, 4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents. This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations. However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment.

Fluorescence and photochemical characterization of phytochromes A and B in transgenic potato expressing Arabidopsis phytochrome B.

Phytochrome in etiolated sprouts of wild type (WT) potato and its transgenic strains (DARA5 and DARA12) expressing Arabidopsis thaliana phytochrome B (phyB) was investigated using low-temperature (85 K) fluorescence spectroscopy and photochemistry. Phytochrome content, [Ptot], position of the Pr emission and excitation spectra, lambda(max), and extent of the Pr-->lumi-R, gamma1, and Pr-->Pfr, gamma2, phototransformations (at 85 and 273 K, respectively) were shown to vary in the transgenic lines and WT depending on tissue used (upper vs. lower parts of etiolated sprouts) and light-induced phytochrome depletion. Differences in the parameters between the transgenic lines and WT were detected which were interpreted in terms of the two phenomenological Pr types: a labile Pr' with gamma1 approximately 0.5 consisting of a major phytochrome A (phyA) fraction (phyA') and a relatively conserved Pr" with gamma1 = 0 comprising a minor phyA fraction (phyA") and phyB. Both DARA lines had higher [Pr"] as compared with WT in the lower parts of etiolated stems, especially after light-induced phytochrome depletion (residual phytochrome in DARA5 and DARA12 made up to one-third of its initial level vs. <5% in WT). These differences were associated with the expression of Arabidopsis phyB in the DARA lines and its higher light stability than that of phyA. Arabidopsis phyB expressed in potato was characterised by lambda(max) = 683/669 nm in the emission/excitation (absorption) spectra and gamma1 = 0. PhyB also revealed a relatively low gamma2 (approx. 0.5) and its early red drop as compared with the gamma2 wavelength dependence for phyA. This is believed to contribute to the lower signalling ability of phyB and to confine the region (red) of its physiological activity.

Technical advance: transcriptional activator TGV mediates dexamethasone-inducible and tetracycline-inactivatable gene expression

A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven PTop10 reach from undetectable levels of the reporter enzyme beta-glucuronidase in the absence of dexa- methasone to induced levels reaching 15-20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.

Heterologous expression of Arabidopsis phytochrome B in transgenic potato influences photosynthetic performance and tuber development.

Transgenic potato (Solanum tuberosum) plants expressing Arabidopsis phytochrome B were characterized morphologically and physiologically under white light in a greenhouse to explore their potential for improved photosynthesis and higher tuber yields. As expected, overexpression of functional phytochrome B caused pleiotropic effects such as semidwarfism, decreased apical dominance, a higher number of smaller but thicker leaves, and increased pigmentation. Because of increased numbers of chloroplasts in elongated palisade cells, photosynthesis per leaf area and in each individual plant increased. In addition, photosynthesis was less sensitive to photoinactivation under prolonged light stress. The beginning of senescence was not delayed, but deceleration of chlorophyll degradation extended the lifetime of photosynthetically active plants. Both the higher photosynthetic performance and the longer lifespan of the transgenic plants allowed greater biomass production, resulting in extended underground organs with increased tuber yields.

Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation.

Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr from initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.

Ferredoxin-1 mRNA is destabilized by changes in photosynthetic electron transport.

In transgenic tobacco, pea Ferredoxin-1 (Fed-1) mRNA accumulates rapidly in response to photosynthesis even when the transgene is driven by a constitutive promoter. To investigate the role of photosynthesis on Fed-1 mRNA stability, we used the tetracycline repressible Top10 promoter system to specifically shut off transcription of the Fed-1 transgene. The Fed-1 mRNA has a half-life of approximately 2.4 hr in the light and a half-life of only 1.2 hr in the dark or in the presence of the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These data indicate that cessation of photosynthesis, either by darkness or DCMU results in a destabilization of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is reduced immediately upon transfer to darkness, suggesting that Fed-1 mRNA destabilization is a primary response to photosynthesis rather than a secondary response to long-term dark adaptation. Finally, the two different methods for efficient tetracycline delivery reported here generally should be useful for half-life measurements of other mRNAs in whole plants.

CHEMICAL CONTROL OF GENE EXPRESSION.

Promoters that respond to otherwise inactive chemicals will enhance the tools available for analyzing gene function in vivo and for altering defined traits of plants at will. Approaches to provide such tools have yielded plant promoters that respond to compounds activating defense genes. In addition, the transfer of regulatory elements from prokaryotes, insects, and mammals has opened new avenues to construct chemically inducible promoters that respond to signals normally not recognized by plants. This review describes results and applications of these two approaches.

Recombinant type A and B phytochromes from potato. Transient absorption spectroscopy.

The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.

Oligogalacturonides Prevent Rhizogenesis in rolB-Transformed Tobacco Explants by Inhibiting Auxin-Induced Expression of the rolB Gene.

Oligogalacturonides elicit several defense responses and regulate different aspects of growth and development in plants. Many of the development-related effects of oligogalacturonides appear to be amenable to an auxin antagonist activity of these oligosaccharins. To clarify the role of oligogalacturonides in antagonizing auxin, we analyzed their effect on root formation in leaf explants of tobacco harboring the plant oncogene rolB. We show here that oligogalacturonides are capable of inhibiting root morphogenesis driven by rolB in transgenic leaf explants when this process requires exogenous auxin. Because rolB expression is induced by auxin and dramatically alters the response to this hormone in transformed plant cells, the inhibiting effect of oligogalacturonides could be exerted on the induction of rolB and/or at some other auxin-requiring step(s) in rhizogenesis. We show that oligogalacturonides antagonize auxin primarily because they strongly inhibit auxin-regulated transcriptional activation of a rolB-[beta]-glucuronidase gene fusion in both leaf explants and cultured leaf protoplasts. In contrast, oligogalacturonides do not inhibit rhizogenesis when rolB transcriptional activation is made independent of auxin, as shown by the lack of inhibition of root formation in leaf explants containing rolB driven by a tetracycline-inducible promoter.

Tetracycline-regulated reporter gene expression in the moss Physcomitrella patens.

As ancestors of higher plants, mosses offer advantages as simple model organisms in studying complex processes such as development and signal transduction. Overexpression of transgenes after genetic transformation is a powerful technique in such studies. To establish a controllable expression system for this experimental approach we expressed a chimeric protein consisting of the Tn1O-encoded Tet repressor and the activation domain of Herpes simplex virion protein 16 in the moss Physcomitrella patens. We showed that this protein activates transcription from a suitable target promoter (Top 1O) containing seven operators upstream of a TATA box. In media containing very low levels of tetracycline (1 mg/l), expression levels of a beta-glucuronidase (GUS) reporter gene dropped to <1% of that in the absence of tetracycline. This regulation is due to interference of tetracycline with the DNA binding activity of the Tet repressor portion of the chimeric transcriptional activator. Stable transformants grown for three weeks on tetracycline-containing media showed negligible GUS activity, whereas GUS was expressed strongly within 24 h of transfer to tetracycline-free media. Potent and stringently regulated expression of other, physiologically active genes is thus readily available in the moss system using the convenient ToplO expression system.

Function of phytochrome A in potato plants as revealed through the study of transgenic plants.

We have generated transgenic potato plants (Solanum tuberosum) containing the potato phytochrome protein encoded by the PHYA gene cDNA (phyA) in sense or antisense orientation under the control of the 35S cauliflower mosaic virus promoter. Plants with increased and decreased phyA levels were analyzed. When grown under white light, development and growth of sprouts and plants were barely distinguishable from wild type. Under continuous far-red light, stem extension, leaf expansion, and hook opening of sprouts were accelerated in phyA overexpressors and delayed in antisense plants. Sprouts with reduced phyA levels were less sensitive to red light with regard to stem extension and expression of the small subunit genes for ribulose bisphosphate carboxylase. Under low red light:far-red light ratios, increased phyA levels reduced the stem extension component of the shade-avoidance response, whereas decreased levels led to an increase in the response.

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.

A dominant negative mutant of PG13 suppresses transcription from a cauliflower mosaic virus 35S truncated promoter in transgenic tobacco plants.

TGA1a and PG13 constitute a family of tobacco basic leucine zipper (bZIP) proteins that bind to activating sequence-1 (as-1), which is one of the multiple regulatory cis elements of the cauliflower mosaic virus (CaMV) 35S promoter. After truncation of the CaMV 35S promoter down to position -90 (CaMV 35S [-90] promoter), transcription stringently depends on the presence of as-1, which is recognized by nuclear DNA binding proteins called ASF-1. The role of the TGA1a/PG13 bZIP family in the formation of ASF-1 and in transcriptional activation of the CaMV 35S (-90) promoter has not yet been demonstrated in vivo. We constructed transgenic tobacco plants expressing a mutant of potato PG13, which lacks its wild-type DNA binding domain. This mutant acts as a trans-dominant inhibitor of ASF-1 formation and of expression from the CaMV 35S (-90) promoter, showing that PG13 can specifically interact with proteins necessary for these processes. Although we did not observe any other obvious phenotypic changes, these transgenic plants are a potentially valuable tool in identifying whether TGA1a and PG13 are involved in controlling promoters encoded in the plant genome.